A melanopsin ganglion cell subtype forms a dorsal retinal mosaic projecting to the supraoptic nucleus

Visual input to the hypothalamus from intrinsically photosensitive retinal ganglion cells (ipRGCs) influences several functions including circadian entrainment, body temperature, and sleep. ipRGCs also project to nuclei such as the supraoptic nucleus (SON), which is involved in systemic fluid homeostasis, maternal behavior, social behaviors, and appetite. However, little is known about the SON-projecting ipRGCs or their relationship to well-characterized ipRGC subtypes. Using a GlyT2Cre mouse line, we show a subtype of ipRGCs restricted to the dorsal retina that selectively projects to the SON. These ipRGCs tile a dorsal region of the retina, forming a substrate for encoding ground luminance. Optogenetic activation of their axons demonstrates they release the neurotransmitter glutamate in multiple regions, including the suprachiasmatic nucleus (SCN) and SON. Our results challenge the idea that ipRGC dendrites overlap to optimize photon capture and suggests non-image forming vision operates to sample local regions of the visual field to influence diverse behaviors.


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Replication Randomization Blinding All data generated or analyzed during this study are included in this published article (and its supplementary information files). Source data are provided with this paper.
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No statistical methods were used to predetermine sample sizes and sample sizes arose by using the minimum number of animals to produce a statistically significant and reproducible result.
For animals crossed with a fluorescent reporter, no data was excluded. For brain injections into the SON, n = 2 animals with minimal retrolabeled cells in the retina, were excluded from the analysis. Given the depth and size of the SON we attribute this to a failed central injection. Brain recordings were excluded if they did not produce a photo-inducible current. In the SCN recordings: n = 23 cells were excluded. In the IGL, n = 53 cells were excluded. In the SON, n = 12 cells were excluded. For the stereotactic eye injections 9 animals were unilaterally injected. 6 animals exhibit dense expression and brains were collected and sectioned. The other 3 animals were excluded because they exhibited poor eye expression and would make central projections challenging to interpret. Brain sections were cut at 200um and brain regions were localized using DAPI staining, immuno-staining of arginine vasopressin, and referenced to the Franklin & Paxinos Mouse Brain Coordinate Atlas. Brain slices where structures could not be confidently identified, were damaged, or were sectioned irregularly (given the 200um thickness) were not included in the quantification (SCN & SON; N=2/6 not included. OPN; N = 1/6 not included.
Blinding was not possible in our study due to the need to confirm genotype to aid in experimental feasibility of electrophysiological experiments and the requirement for the genotype to be unmasked for the analysis of our datasets. For example many of the data can not be analyzed with masking as we count fluorescent cell bodies or axons where fluorescence is driven by Cre and the presence of fluorescence Validation reveals the genotype. We could not blind the application of experimental drugs in pharmacological bath applications due to the extremely low hit-rate in our experiments (for example photoresponses in the SCN were obtained from 11 total cells from over 100 recorded cells). Rabbit anti-melanopsin (Advanced targeting systemsl cat AB-n39) is an affinity-purified version of the n-38 antibody (RRID: AB_1608077) and has been previously published in 11 scientific articles first by Dumitrescu et. al. 2009 who state that it "is raised against a synthetic peptide consisting of the 15 N-terminal amino acids of mouse melanopsin (MDSPSGPRVLSSLTQ). It produces no staining in melanopsin knockout mice (Opn4 !/!)." Using the OPN4Cre mouse line we also validated this antibody produces no staining in OPN4Cre+/+ mice, which are OPN4-/-or knockout mice (Chen et. al. 2011). Chicken anti-mCherry antibody (Abcam ab205402; RRID AB2722769) is raised against the recombinant protein (His-tag) corresponding to mCherry (sequence from Shaner NC et al. Nature Biotechnology 22:1567-1572(2004. This antibody has been published in 37 articles and stains a band with the predicted molecular weight of 30kDa in a lysate of HEK293 cells transfected with pFin-EF1-mCherry vector. Goat anti-GFP antibody (ab5450; RRID AB_304897) is a polyclonal antibody raised against the recombinant full length protein corresponding to GFP. This antibody produces signal amplification in cells expressing GFP and has been references in 10 publications. Chicken anti-GFP (Aves labs GFP-1020; RRID AB_2307313) is a polyclonal antibody raised against recombinant GFP expressed in Escherichia coli. Antibodies were analyzed by western blot analysis (1:5000 dilution) and immunohistochemistry (1:500 dilution) using transgenic mice expressing the GFP gene product. Western blots were performed using BlokHen® (Aves Labs) as the blocking reagent, and HRP-labeled goat antichicken antibodies (Aves Labs, Cat. #H-1004) as the detection reagent. The vendor lists this antibody as being referenced in 643 research articles. In our experiments, this antibody strongly amplifies the signal that is produced in mice where Cre drives the production of EGFP in specific neuronal populations (GlyT2Cre;Ai140; Ai140 from Jackson mouse line 030220). Goat anti-cholera toxin subunit B (List Labs -Cat#: 703; RRID AB_10013220) is a goat polyclonal antibody against the Cholera Toxin B subunit and has been cited in 137 research articles. In our experiments, this antibody produces strong amplification of the axon-terminals of retinal ganglion cells in the suprachiasmatic nucleus following eye injections of Cholera Toxin B subunit, as previously published in other papers using the same methods (Hattar et al. 2006 PMID: 16736474) and when conjugated to a secondary antibody with a far-red (Alexa 647) fluorophore. Rabbit anti-vasopressin antibody (lmmunostar -Cat#: 20069; RRID AB_572219) is a rabbit polyclonal antibody against argenine vasopressin and has 67 linked citations. The antibody produces significant fluorescent staining and significant biotin-avidin/HRP staining at a 1/2,000 -1/4,000 dilution in rat hypothalamus. Staining is completely eliminated by pretreatment of the diluted antibody with 10 µg/mL of arginine vasopressin. Preadsorption with as much as 100 ug/mL of oxytocin had no effect on immunolabeling, confirming specificity. In our experiments this antibody labeled neurons in the suprachiasmatic nucleus, the paraventricular nucleus, and the supraoptic nucleus as published in previous studies (PMID: 34561434; PMID: 21525287; PMID: 30283813). Rabbit anti-opsin antibody blue (Millipore Sigma -Cat#: ab5407; RRID AB_177457) is a rabbit polyclonal antibody against recombinant human blue opsin. Validation information can be found on the manufacturers website: https://www.emdmillipore.com/US/en/product/Anti-Opsin-Antibody-blue,MM_NF-AB5407. In our experiments in mice this antibody stained S-cone photoreceptor outer segments located predominantly in the ventral retina as previously published (PMID: 15930394; PMID: 18076080). Mouse anti-Neurofilament H (SMI-32 Biolegend; previously Covance Cat SMI32;RRID AB_509997) is a mouse affinity purified monoclonal antibody against neurofilament H and has been referenced in 146 publications. Validation information can be found at the manufacturer's website: https://www.biolegend.com/en-us/products/ purified-anti-neurofilament-h-nf-h-nonphosphorylated-antibody-11475. In our experiments SMI-32 labeled alpha-type retinal ganglion cells as previously published (PMID: 30017393; PMID: 24440397). Guinea Pig anti RBPMS (Phosphosolutions Cat# 1832-RBPMS lot NB322g) is a polyclonal antibody raised against a synthetic peptide corresponding to amino acid residues from the N-